This proposal seeks to study the distribution, mechanism, and regulation of H/HCO3 transport in the rat inner medullary collecting duct (IMCD). Previous studies have established that the IMCD is a major site of urinary acidification and net acid secretion. Recent morphologic and functional studies have extensively examined the mechanisms and regulation of H/HCO3 transport in the cortical and outer medullary collecting duct. The IMCD has not been studied previously by in vitro microperfusion because of its technical difficulty. Due to a number of recent technical advances, it is now possible to dissect the IMCD from germ-free animals. We will isolate and perfuse the IMCD in vitro permitting us to (1) determine the axial distribution of H/HCO3 transport along the IMCD; (2) identify the mechanism of transport using fluorescent measurements of cell pH; (3) identify the cell types which secrete protons with fluorescent and optical microscopy; and (4) determine the mechanism of regulation of transepithelial transport using all the above techniques. These studies will supply insights into the pathway which mediates proton secretion in the terminal portion of the nephron and will provide a better understanding of the role of the IMCD in renal acid-base balance. During my postdoctoral training at the NIH with Dr. Maurice Burg, I learned the techniques of in vitro microperfusion and fluorescence microscopy, and I used these to examine the mechanisms of HCO3 and ammonia transport in the proximal tubule and collecting duct segments. These techniques have been successfully transferred to my new laboratory at Southwestern Medical School. I will now apply these techniques (along with a more sensitive assay for total CO2 which I developed specially for these studies), to address the above problems in the rat inner medullary collecting duct. My sponsor, Dr. Robert Alpern, has extensive experience performing similar studies in the proximal tubule and will be involved extensively in the supervision of these studies. In addition, Dr. Donald Seldin, who has a longstanding interest in distal acidification, will also be involved.